1. Field of the Invention
The invention is directed to a new class of immune stimulating peptides. In particular, the invention is directed to methods of obtaining these peptides from mammalian serum and to methods of administering these peptides for the therapeutic or prophylactic treatment of viral and infectious diseases and cancer.
2. Description of the Background
Discovering agents that potentiate the immune response is a driving force in modern drug research. Bioactive peptides, such as cytokines, chemokines, and cationic peptides, are classes of “relatively” low molecular weight compounds that have shown promise in this area of research. At least nine immuno-defense peptide products are commercially available with annual sales of over $4 billion (Latham, 1999).
The concept of immunostimulation originated in 1907, when William B. Coley noted spontaneous tumor regression in some patients after an episode of septicemia (Rush and Flaminio, 2000). In human and veterinary medicine, immunostimulant preparations are used primarily for treatment of chronic viral or bacterial infections. In some instances, immunostimulants have demonstrated efficacy as primary or adjunct treatment of neoplastic conditions (Rush, 2001). The proposed mechanism of action of nonspecific immunostimulant preparations is macrophage activation and subsequent release of cytokines that enhance the immune response (Rush and Flaminio, 2000). Prophylactic administration of immunostimulant preparations prior to pathogen exposure can decrease morbidity and mortality associated with acute infection (Rush, 2001).
In human medicine, immunoregulator preparations have progressed from crude microbial, viral, plant, and thymic extracts to synthetic viral complexes and chemically defined drugs (e.g., recombinant cytokines). The crude extract preparations induce nonspecific immunoregulatory activity via a generalized macrophage activation. The new generation of immunoregulators, such as the recombinant cytokines, have selective effects on particular components of the immune system (Rush, 2001).
Because cells of the immune system circulate through the blood and lymphatic system, serum is a logical place to look for immunoregulators. To date, only about half of the over 100 serum proteins have been isolated and characterized (de Gruyter, 1997), leaving a variety of proteins and peptides as potential immunoregulators. For example, Caprine Serum Fraction Immunomodulator (CSFI) is a non-adjuvanted immunostimulant derived from goat serum (Ansley, Daniel R. Composition and Method for Immunostimulation in Mammals, U.S. Pat. No. 5,219,578; Jun. 15, 1993). CSFI is ill-defined by Ansley but is said to be composed of a mixture of serum proteins and peptides, 67% of which is immunoglobulin. In that patent Ansley describes a method for collecting the immunoglobulin containing fraction of goat serum from non-immunogenicly challenged goats. That process consisted of precipitation with sodium sulfate followed by dialysis of the re-suspended precipitate in a 30,000 dalton MW cut-off dialysis membrane to remove salts and other low molecular weight substances. The dialyzed fraction was then shown to be efficacious in the treatment of a wide variety of animal diseases including equine lower respiratory disease, ovine footrot, bovine shipping fever, bovine respiratory disease, canine lymphoma, bovine lymphoma, and canine parvovirus.
Hamm recently established evidence that this immunoglobulin containing fraction of goat serum could be used as an adjunct to conventional antibiotics in the treatment of equine lower respiratory disease. In that study more than twice as many horses (86%) were able to recover in a three week period when the treatment was augmented with the caprine serum fraction as compared to the control group (35%) that only received antibiotic treatment (Hamm, 2002). This fraction is now marketed in the U.S. under the trade name PulmoClear™ for the treatment of equine lower respiratory disease.
Some immunoregulators derived from one species appear to provide a short-term immunity from pathogenic infections when administered to a different host species. In a recent study, Willeford was able to establish that a fraction of caprine serum, substantially free of immunoglobulins, could confer significant protection to chickens challenged with a lethal dose of Pasteurella multocida when the caprine serum fraction was administered 24 hours prior to the bacterial challenge (Willeford, 2000). Similar results were noted by Parker in mice challenged with Salmonella typhimurium (Parker, 2002).
Immunoregulators have been derived from sources other than animal serum as well. For instance, a variety of immuno-stimulants have been derived from mycobacterial products (Ford, 1986; Werner and Zerial, 1984; Diasio and LoBuglio, 1995). Regressin-V, an emulsion of mycobacterial cell wall fragments, is licensed for the treatment of a variety of neoplasias in animals. A killed suspension of Propiobacterium acnes, Immunoregulin, is licensed for veterinary use in advanced neoplasia as an adjunct to other therapies. Although these products are capable of stimulating the immune system in animals in a non-specific manner and are therapeutically efficacious, they have also been observed to initiate untoward effects such as fever and allergic reactions that arise from the broad spectrum of the immune stimulating action (Kruth, 1998).
Immunoregulators can be divided into three main groups: (a) immuno-suppressive agents; (b) immuno-stimulating agents (e.g., bacillus Calmette-Guérin vaccine); and (c) the remaining immunoregulators, which include biological response modifiers (e.g., colony stimulating factors, interleukins, interferons, and tumor necrosis factors) (Takx-Kohlen, 1992; Molloy et al., 1993). Cytokines are soluble, low molecular weight polypeptides and glycopepetides produced by a broad range of cell types that have suppressive or enhancing effects on cellular proliferation, differentiation, activation, and motility. For the most part, they are not constitutively secreted, but are produced in response to stimulation by infectious agents or their derived products (e.g., endotoxin), inflammatory mediators, mechanical injuries, and cytokines themselves (Kogut, 2000).
Interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and interferon (IFN) are three cytokines that participate in the immune response. IL-1 is involved in the host's response to antigenic challenge and tissue injury, and has been shown to increase the resistance of mice to pathogenic organisms such as Listeria, Escherichia coli, and Candida albicans (Czuprynski and Brown, 1987; Cross et al., 1989; Pecyk et al., 1989). TNF-α and δ-IFN increased the resistance of mice to Salmonella typhimurium (Morrissey and Charrier, 1994). Human δ-IFNs have potent antiviral and antiproliferative activities, and are utilized as anticancer and antiviral therapeutic agents (Chang et al., 1999).
Three families of low molecular weight peptides that have immune regulatory properties are the tachykinins, the thymic hormones, and cationic peptides. The tachykinins are a family of closely related short neuropeptides that were initially identified by their activities as neurotransmitters. Tachykinins are now known to mediate such diverse activities as the proliferation of T-cells, release of TFN-γ, TNF-α, IL-1 and IL-6, and enhanced secretion of immunoglobulins (Maggio, 1990; Eglezos et al., 1991).
The thymic hormones are a family of proteins and peptides whose exact biological role is unknown. They are known to participate in the regulation and differentiation of thymus-derived lymphocytes and have been shown to act like cytokines. Some thymic hormones have been shown to reconstitute defective cell-mediated immunity in patients with various neoplastic diseases and secondary immune deficiencies as a result of chemo- and/or radiotherapy (thymic humoral factor) as well as enhance the production of IL-1, IL-2, IFN-γ and TNF-α (thymosin fraction 5) (Cohen et al., 1979; Dardenne and Savino, 1990).
Some cationic peptides have been observed to initiate an immunostimulant response. A decameric peptide was shown to impede the growth and spreading of established tumors (Folkman, 1999). Other peptides promote antibacterial, antifungal, antiviral, and even wound healing properties (Sanglier et al., 1993; Mizuno et al., 1995; Hancock, 1999). It is believed that these “defense” peptides are more general in their actions than antibodies, and as such, have a broader range of activity (Hancock, 1999).